Polyketide natural products are structurally complex molecules produced by a variety of organisms and serve an important role in medicine, most often utilized as antibiotic and anticancer agents. There is great interest in developing new chemotherapeutic agents due to the toxic side effects of many currently utilized therapies; additionally, there is an urgent need to develop new antibiotics due to the rapid emergence of antibiotic-resistant pathogens (e.g. methicillin-resistant Staphylococcus aureus, MRSA). Because of their impressive biological activity and the challenge of accessing polyketides through chemical synthesis, there has been extensive research in genetically reprogramming the biosynthetic pathways of the organisms that produce these natural products, and numerous analogs have been synthesized by this approach. In order to further expand the diversity of biologically active compounds that can be accessed by synthetic biology, site-selective modification of polyketides must be developed. One approach to functionalize polyketides is to enzymatically modify the polyketide during the process of biosynthesis (while the substrate remains bound to an acyl carrier protein (ACP)). The acyl carrier protein could 1) deliver the substrate directly to the modifying enzyme, and 2) facilitate the transformation by lowering the energy barrier through favorable protein-protein interactions. Oxygenation of polyketides can have a dramatic impact on their biologically activity, and cytochrome P450 monooxygenases often act to site-selectively oxidize polyketide macrolides following their biosynthesis. There are several reports, however, of cytochrome P450 enzymes that act on substrates bound to an ACP. For example, BioI is a cytochrome P450 monooxygenase involved in biotin biosynthesis that is known to oxidize ACP-bound fatty acids at the C7/C8 positions. We propose to use BioI as a platform for engineering site-selective C-H oxidation of polyketides. To achieve this goal, we will study the Michaelis-Menten kinetics of BioI oxidation in order to determine whether the ACP facilitates the C-H oxidation (by lowering Km and increasing the kcat of the oxidation). In addition, we will examine the substrate specificity for BioI and determine whether the ACP will allow non-native substrates to undergo oxidation. Finally, we will incorporate BioI into module 2 of the nanchangmycin synthase (NANS) and erythromycin synthase (DEBS). We will first establish whether BioI is capable of oxidizing polyketides bound to isolated ACPs, and we will then co-express BioI along with NANS and DEBS, and determine whether BioI can oxidize ACP bound polyketides that are generated on a polyketide synthase assembly line. We will use these ACP-P450 systems to engineer site-selective C-H oxidation of polyketides, which will expand the diversity of biologically active macrolides accessible through biotic synthesis.